Downregulation of Circulating Long Non-Coding Rnas GHRLOS and LINC00852 Associated with Type 2 Diabetes Mellitus

Research Article

J Dis Markers. 2020; 5(1): 1038.

Downregulation of Circulating Long Non-Coding Rnas GHRLOS and LINC00852 Associated with Type 2 Diabetes Mellitus

Al-Harithy R* and Anbari D

Department of Biochemistry, King AbdulAziz University, Saudi Arabia

*Corresponding author: Al-Harithy R, Biochemistry Department, King AbdulAziz University, Saudi Arabia

Received: September 08, 2020; Accepted: October 20, 2020; Published: October 27, 2020


Long Non-Coding Rnas (lncRNAs) have an important role in many biological processes that are associated with several chronic diseases including Type 2 Diabetes Mellitus (T2DM). The aim of the present study is to identify novel biomarkers for T2DM by investigating the differentially expressed circulating lncRNAs, which are transcribed from Ghrelin (GHRL) Gene Region. Bioinformatic analysis was used to identify lncRNAs-GHRL candidates. The expression levels of the selected lncRNAs-GHRL were determined using Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) assay on 62 diabetic patients and 32 non-diabetic controls. Receiver Operating Characteristic (ROC) curve was used to assess the discriminatory power of the candidate lncRNAs as biomarkers for T2DM. The expression profiles demonstrated that lncRNA GHRLOS and LINC00852 were significantly downregulated in the diabetic patients compared to the non-diabetic controls (P < 0.0001). The value of the Area Under The Curve (AUC) for GHRLOS and LINC00852 were 0.98 at a cut-off point of 1.03 and 0.96 at a cut-off value of 1.19; respectively. In conclusion, this study revealed that lncRNA GHRLOS and LINC00852 are novel biomarkers associated with T2DM and they might have regulatory roles in the development of the disease.

Keywords: Ghrelin (GHRL) gene; Long non-coding RNA; GHRLOS; LINC00852; Type 2 Diabetes Mellitus; Biomarker


LncRNA: Long Non-Coding RNA; T2DM: Type 2 Diabetes Mellitus; GHRL: Ghrelin Gene; qRT-PCR: Quantitative Real Time Polymerase Chain Reaction; ROC: Receiver Operating Characteristic; GHRLOS: Ghrelin Gene Opposite Strand; AUC: Area Under The Curve; ADA : American Diabetes Association; HbA1c: Hemoglobin A1c; cDNA: Complementary DNA; GAPDH: Glyceraldehyde 3-Phosphate Dehydrogenase; BMI: Body Mass Index; NCD: Noncommunicable Diseases; MAPK: Mitogen-Activated Protein Kinase; S100A9:S100 CalciumBinding Protein A9; JNK: C-Jun N-Terminal Kinase; PI3K: Phosphatidylinositol 3-Kinase; Protein Kinase-Uncoupling Protein 2 (AMPK-UCP2)


Type 2 Diabetes Mellitus (T2DM) is a complex metabolic disease characterized by insufficient insulin production and/or insulin resistance of human tissues [1,2]. It is a worldwide health crisis that leads to development of life-threatening health complications such as retinopathy, nephropathy, and neuropathy [3]. Despite of the great efforts that have been achieved in diagnosing and treating T2DM patients, the exact mechanism of its development remains poorly understood. Several investigations have been conducted to understand the pathogenesis of T2DM; however, the huge role that epigenetics may play in T2DM remains small compared to that devoted to traditional genetics work [4].

Epigenetic biomarkers have been identified and Long Noncoding Rnas (lncRNAs) are important regulators of the epigenetic status of the human genome. They are a type of Non-Coding Rnas (ncRNAs) which exceed 200 nucleotides in length without protein-coding potential [5,6]. The biogenesis process of lncRNAs is mainly similar to mRNA which is mediated by RNA polymerase II [7,8]. Moreover, different isoforms of lncRNAs can be transcribed from the same locus as a result of alternative splicing, cleavage and polyadenylation [9]. According to lncRNAs’ roles and biological positions, they can be classified as sense, antisense, intergenic and intronic lncRNAs [10]. The importance of lncRNAs are known in diverse of biological processes including cell’s functions, metabolism and genomic regulations [11,12]. The regulatory role may include the ability of lncRNAs to bind with Transcriptional Factors (TFs) and / or RNA polymerase II to transcription process. Also, they can indirectly regulate the expression level of mRNA by affecting its splicing, transporting and translation [5]. In addition, lncRNAs have been reported to contribute in genomic methylation and chromatin remodeling processes to regulate gene expression level [13]. Moreover, the presence of some genetic variation such as Single Nucleotide Polymorphisms (SNPs) within lncRNAs may contribute in modifying their secondary structure and altering their expression patterns. This would affect their regulatory role and consequently, contributing in the progression of the diseases [14]. To date, numerous studies have attempted to explain the association between lncRNAs and the development of various diseases [15,16]. However, the molecular mechanisms of lncRNAs in glucose homeostasis and T2DM remain elusive [17- 19]. The hunger hormone ghrelin or “lenomorelin” is a circulating peptide hormone which is secreted mainly from the stomach, while trace amounts derived from the pancreas [20,21]. Ghrelin is involved in several biological functions, which include body weight regulation, blood glucose homeostasis, appetite stimulation and insulin metabolism [22, 23]. Several studies have reported that insulin has inhibitory effect on ghrelin [23,24]. It has been suggested that ghrelin levels and insulin are inversely correlated in healthy human, which indicates the occurrence of feedback inhibition between them. Therefore, circulating ghrelin levels decreases in response of insulin secretion [25,26]. Previous studies revealed that ghrelin gene is strongly associated with insulin resistance and T2DM [27,28]. Thus, more Genetic and Epigenetic Studies on Ghrelin (GHRL) Gene are needed to understand its regulatory role in T2DM. The aim of this study is to identify potential circulating lncRNA-GHRL biomarkers that play a role in the development of T2DM.

Materials and Methods

Patients and Healthy Controls: This study was approved by the National Committee of Bio and Med. Ethics at King Abdul-Aziz University and Medical College, Jeddah, Saudi Arabia (reference number: HA-02-J-008). Between March 2019 and October 2019, patients with T2DM were recruited from the Diabetic and Endocrine Care Centre in Jeddah, Saudi Arabia. All participants provided informed consent prior to enrolment. All patients with T2DM were diagnosed according to the diagnostic criteria by the American Diabetes Association (ADA). All participants were Saudi, aged 40 years or older. For the diabetic patients, they were all diagnosed as diabetics since at least two years and they were taking diabetic medications such as metformin and insulin. For the non-diabetic controls, HbA1c level was between 4.8% and 5.6% and they were not under any medical conditions. Subject with independent chronic or acute medical conditions independent of diabetes and its complications were excluded from this study.

Blood Samples and Total RNA Extraction: Peripheral blood samples (5-8 ml) were obtained from each subject. The samples were collected in EDTA tubes. Total RNAs were isolated from whole blood samples using QIAamp RNA blood mini kit (Qiagen, USA) following the manufacturer’s instruction. RNase decontamination solution (RNaseZap) was used to prevent the degradation of RNA. The RNA concentrations were measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Bioinformatics Analysis And Complementary DNA Synthesis(cDNA): LINCipedia database ( was used to determine lncRNAs that are related to GHRL gene. The analysis using LINCipedia database showed 11 GHRL-associated lncRNAs. For the cDNA synthesis, total RNA samples were reverse transcribed into cDNA using the total transcriptome cDNA synthesis kit (abm, Canada). All cDNA samples were diluted in 100 μl nucleasefree water and were stored at -20 °C prior to use.

Quantitative Real Time Polymerase Chain Reaction (Qrt-PCR) Analysis: Complementary Dna (cDNA) was amplified via qRTPCR using PowerUp Syber Green Master Mix kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Primers were purchased from Macrogen, Inc (Table 1). The thermocycling protocol included the following steps: initial incubation at 50ºC for 2 minutes, followed by 40 cycles amplification at 95ºC for 15 seconds, annealing at 55-60°C for 15 seconds, and extension at 72ºC for 1 minutes. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used as an endogenous control to normalize the lncRNAs expression levels. All reactions were performed in triplicate.